The present invention relates to detection of ligand-receptor interactions by monitoring changes in refractive index and in particular to detection of such interactions between relatively small ligands and relatively large receptors.
Refractive index is defined as the ratio of the velocity of a specific radiation in a vacuum to its velocity in a given medium. The direction of a ray of light is changed (i.e., refracted) upon passage from one medium to another of different density or when traversing a medium whose density is not uniform. One method of monitoring such refraction is Surface Plasmon Resonance (SPR), which is based upon the phenomenon of total internal reflection, wherein light traveling through a medium of higher refractive index (e.g., glass) is totally internally reflected upon encountering a medium of lower refractive index (e.g., solution) at a sufficiently oblique angle. In SPR detection, the intensity of the reflected light is dampened by the presence of a metal surface at the interface of the two media. The decrease in intensity occurs at a well-defined angle, which is dependent on the refractive indices of the two media, referred to as the xe2x80x9cresonance anglexe2x80x9d. As proteins adsorb onto the metal surface, the refractive index of the solution near the interface changes, shifting the angle at which the reflected light is dampened (i.e, shifting the resonance angle). Detection of the shift in the resonance angle is the basis of an apparatus called the BIACORE(trademark), in which one ligand is immobilized onto a chemically modified gold surface, and the association and dissociation of a soluble ligand to the immobilized ligand is monitored as a function of real time. This optically based sensor allows direct detection of proteins in complex solutions, such as fermentation media (H. V. Hsieh, et al., 1998. Vaccine 16, 997-1003). The signal output of the BIACORE(trademark) is referred to as a resonance unit (RU) and reflects the resonance angle. In general for proteins, 1 RU is approximately 1 pg/mm2. Other instruments for measurement of SPR are available from Bio-Tul (Plasmoon) and Texas Instruments (Spreeta). Other companies developing instruments based on surface refractive index changes include Luna Innovations (Long Period Grating-based sensors, or LPG) and Affinity Sensors (IAsys).
SPR and similar techniques have been used to analyze the binding of ligands and receptors. Because the refractive index of the solution near the solid-liquid interface can be affected by the mass of the molecules at the interface, in general binding of a ligand and receptor increases mass and results in an increase in the angle of reflection. SPR instruments such as the BIACORE(trademark) monitor the binding event continuously. Using these techniques, binding kinetics and affinity can be analyzed.
More recently SPR has been used to study conformational changes in proteins. H. Sota and Y. Hasegawa (1998. Anal. Chem. 70, 2019-2024) immobilized E. coli dihydrofolate reductase and observed an increase in SPR signal with acid denaturation of the protein. S. Boussaad, et al. (2000. Anal. Chem. 72, 222-226) reported the use of SPR to monitor the electronic state of cytochrome c and observed a decrease in resonance angle as the protein was switched from the oxidized to the reduced state, indicating an associated conformational change. These examples, however, do not involve receptor-ligand binding.
One shortcoming of SPR analysis of ligand/receptor interactions is that the change in mass upon binding must be large enough to be detected as a change in refractive index. SPR is typically used for monitoring binding of relatively large ligands to immobilized receptors (e.g., antibody/antigen binding). This binding is accompanied by a relatively large increase in signal because the significant increase in mass of the complex results in a large change in refractive index. Although instruments such as the BIACORE(trademark) are quite sensitive, binding of very small ligands to the surface is typically not detectable because the increase in mass is too small to be detected as an increase in refractive index. An example of a binding reaction which would be expected to be below the detection limits of currently available SPR instruments (based on the minimal increase in mass upon binding) is binding of calcium to calmodulin (CaM). For this reason, alternative assays such as the fluorescence assay described by T. L. Blair, et al. (1994. Anal. Chem. 66,300-302) which do not involve SPR have been used to study conformational changes upon CaM/calcium binding. SPR assays for analysis of CaM binding so far have been limited to systems in which calcium mediates binding of CaM in solution to an immobilized oligopeptide, such as reported by T. Ozawa, et al. (1999. Biochim. Biophys. Acta 1434, 211-220) and E. Takano, et al. (1994. FEBS Lett. 352, 247-250). In these systems the use of CaM (molecular weight) xcx9c17000) as the solution molecule makes the interaction detectable by conventional SPR methods.
Optical fiber long period grating (LPG)-sensors have been used for detection of biological targets by applying affinity coatings to the fiber surface (M. E. Jones, et al. 2000, NSF Design and Manuf. Research Conf., Vancouver, Poster Number SBIR-510). The LPG scatters out light at a particular wavelength based on grating period, fiber refractive index and the refractive index of the surrounding medium. As the affinity coating absorbs the target molecule the refractive index changes and causes a shift in the wavelength of scattered light xe2x80x9cseenxe2x80x9d by the LPG. The authors report detection of xcex2-galactosidase binding to a ligand-based affinity coating (polyclonal antibodies) as a shift in wavelength, however, such binding produces a relatively large change in mass but does not result in a conformational change in the receptor.
The prior art has not reported the use of surface refractive methods such as these for analysis of ligand binding to allosteric receptors. Although CaM/Ca binding is allosteric, the CaM binding studies described above do not detect it because calcium is bound to CaM prior to binding of the complex to the immobilized oligopeptide. Binding of the complex to the oligopeptide, which is the binding being detected, is not allosteric.
It has now unexpectedly been found that changes in refractive index can be used to detect binding of ligands to immobilized receptors when the receptor (e.g., a binding protein) or the receptor-surface complex undergoes a conformational change upon binding to the ligand. The conformational change is detectable even when the ligand is small and the receptor is large, which would not be predicted based on the increase in mass upon ligand binding. Unexpectedly, binding of such allosteric binding agents to their ligands may produce negative deviations in the optical response on SPR (i.e., a decrease in resonance angle). While not wishing to be bound by any particular theory of how the invention operates, Applicants believe that the reduction in signal upon binding may be due to the binding protein xe2x80x9cclosingxe2x80x9d or xe2x80x9ccollapsingxe2x80x9d around the ligand as it binds. This could result in a decrease in the hydrodynamic volume which is greater than the increase in mass upon binding. Conversely, an increase in the optical response has been observed on SPR when the allosteric binding agent xe2x80x9copensxe2x80x9d upon binding of the ligand. This response may result in an increase in the molecular volume due to a conformational change which is more significant than the added mass of the ligand. However, whether the change in refractive index is positive or negative for any given receptor-ligand pair may depend on the method and instrumentation used to make the determination, as the changes observed on LPG do not always follow those observed on SPR.
The present invention therefore provides a novel optical method for detecting and analyzing binding of allosteric receptors to their ligands. The methods of the invention are particularly useful for small ligands which would not be expected to be detectable by changes in surface refractive index because they do not add significant mass upon binding.